These studies represent a continuation of long-term examinations of two biotin-containing enzymes: oxalacetate-methylmalonyl-CoA transcarboxylase from Propionibacterium and pyruvate carboxylase from animal tissues, yeast and Pseudomonas. For transcarboxylase, studies will attempt to characterize the now available crystalline forms of the large form of the enzyme, to characterize the nature of the binding of the biotin-containing small subunit by utilization of various segments of this subunit to bind to the central subunit, to further investigate the role of disulfides in the structure and activity of the enzyme, to characterize the bacterial biotin synthetase, and finally, to look at the distribution of the enzyme in various species of Propionibacterium. Studies with pyruvate carboxylase from Pseudomonas will attempt to elucidate the functional role of the two polypeptides that make up this enzyme by use of monoclonal antibodies and site specific affinity labels. Studies of pyruvate carboxylase in 3T3-Ll cells will attempt to ascertain whether the large observed increased in rate of synthesis of this enzyme which occurs during differentiation is accompanied by a similar increase in mRNA for the protein, whether in vitro translation of mRNA from these cells yield a precursor form of this mitochondrial matrix enzyme and finally, whether post-translational events may be rate-limiting in these cells for the formation of mature, active enzyme.